The genetic code (16) Flashcards
The central dogma
DNA-replication -> RNA (transcription) -> Protein (translation
Represents the flow of genetic information.
How did they know the genetic code had to consists of triplets of nucleotides?
4 different nucleotides were known: A, T, C and G. Since there wasn’t enough variation to code for the 20 amino acids, people suggested that the information was a code consisting of a combination of nucleotides.
With a 2-letter code, there would only be 4x4 = 16 possibilities, which is too few to account for 20 amino acids. Therefore, the code must consist of 3 nucleotides, giving in total 4x4x4 = 64 permutations (kombinasjonsmuligheter).
Marshall Nirenberg’s “polyU experiment” (step-by-step)
- Used a bacterial extract supporting protein synthesis in 20 tubes, each containing only 1 amino acid.
- Added synthetic polynucleotides into each tube. For example, they could add only Us, meaning that the columns must consist of only Us.
- Phenylalanin (Phe) was encoded for by the free Us in the UUU combination.
- Later, similar experiments with other polynucleotides, e.g. triple As and triple Cs, encoding for lysine and proline respectively.
(Should be mentioned in the context of the genetic code’s near universability)
Experiments with synthetic oligos
Allowed to identify the more complicated combinations of bases to amino acids (not only triples). Based on the discovery that trinucleotides can associate with the ribosome, which can recruit amino-charged tRNA.
3 different scenarios:
- Trinucleotide (UUU) and all tRNAs pass through the filter.
- Ribosomes stick to the filter.
- Complex of ribosome, UUU, and Phe-tRNA sticks to the filter (labeled). Confirmed association between triple Us and phenylalanin.
By synthesizing and testing all possible trinucleotides, the researchers were able to match all 20 amino acids with one or more codons.
In what way is the code degenerate?
There are very few amino acids encoded by specific codons (only Met and Trp) - most are encoded by several codons.
Degeneration refers to the property of the genetic code of which a single amino acid can be encoded by multiple codons.
How are the codons decoded? (2 ends of tRNA)
With the help of anticodon-codon interaction by tRNA molecules.
tRNAs are highly structured short RNAs which consist of two ends:
- Acceptor stem, where the tRNA associates with a particular amino acids (tRNA is charged)
- Anticodon, where the decoding of mRNA happens. Consists of 3 residues 5’-3’ which associates with the mRNA codon 3’-5’ (the first nucleotide in the codon associates with the third nucleotide in the anticodon).
The code is degenerate but there are <64 tRNAs. How is this possible? (example)
tRNA molecules consists of an acceptor arm and an anticodon. What defines the anticodon is that the 3 nucleotides are flipped out, placing them in a good position to interact with the nucleotides of the codon.
“Wobble” position of the 1st base: The nucleotide in the first position of the anticodon has the most freedom to move around (flexibility of movement). It allows not only standard interactions with mRNA, but also additional.
Wobble base pairing lets the same tRNA recognize multiple codons for the amino acid it carries. For example can a Phe-charged tRNA with the anticodon 5’-GAA-3’ interact with both 5’-UUC-3’ and 5’-UUU-3’ mRNA codons.
Wobble base pairs
If base are in the THIRD, or wobble, position of codon of an mRNA, then the codon may be recognized by a tRNA having these bases in the FIRST position of the anticodon.
Some examples of wobble base pairs are:
- C with G/I
- A with U/I
- G with C/U
- U with A/G/I
tRNAs containing the base inosine (I) can interact with various nucleotides and thereby several codons.
The 3 principal rules of the genetic code (Syntax)
- Codons are read in the 5’ to 3’ direction.
- Codons are non-overlapping and contain NO gaps.
- The message is translated in a fixed reading-frame, which is set by the initiation codon (methionin).
What does it mean that the genetic code is nearly universal?
Same nucleotides, same rules/syntax.
Some of the codons are encoded differently. You see a variation in the genetic code among species, e.g. is the standard stop codon UGA encoding for an amino acid in certain bacteria. In mitochondria we also see situations where a standard code for an amino acid is used for stop codons.
Which types of mutations can alter the genetic code?
Spontaneous mutations: Result from abnormalities in cellular/biological processes, e.g. errors in DNA-replication.
Induced mutations: Caused by environmental agents, either chemical or physical (incl. infectious agents or radiation). Agents that are known to alter DNA structure are termed mutagens.
Different types of reading-frame mutations
Silent mutation: No change in protein sequence, no consequence. Usually when the mutation happens in the third position (wobble position, flexibility in interaction)
Missense mutation: Change in codon. Could be harmful, depends on where it happens in the protein.
Nonsense mutation: A codon becomes a stop codon. Results in truncated proteins, most often you stimulate nonsense mRNA degradation.
Frameshift insertion: Insertion of a nucleotide. Completely change the amino acid sequence following the mutation site.
Frameshift deletion: Deletion of a nucleotide.
Expanding the genetic code: The principle (why + what does it require?)
If you have a desire to produce proteins with new properties, you can modify the amino acid and manipulate the proteins.
Required are:
- A unique codon
- Orthogonal tRNA
- Orthogonal aminoacyl-tRNA synthethase
(Orthogonal: Something so dissimilar from those occurring in nature that they can only interact with them to a very limited extent)
